Phospholipases A1, A2, C, and D enzymatically cleave the bonds in a diacyl phospholipid as illustrated below: These enzymes have proven useful in the synthetic preparation of a wide variety of phospholipids, sphingolipids, and neutral lipids, and in particular the preparation of new, non-naturally occurring, or non-abundant lipids from more abundant source materials. Similarly, C- and D-type phospholipases which use sphingomyelin as a substrate (sphingomyelin phosphodiesterases), and various glycosidases which use glycolipids as substrates, can be utilized to prepare ceramides and other lipids.
In particular, phospholipase D has been used to convert phosphatidyl cholines (PC) to less common lipids such as phosphatidyl serines (PS) and phosphatidyl glycerols (PG). The reaction is a transesterification (also referred to as transphosphatidylation) between the starting material, PC, and a hydroxyl containing reagent such as glycerol or serine. The enzyme is especially useful in this respect in that it is able to produce the transesterified product in the presence of more than a stoichiometric amount of water (Servi). An organic solvent or detergent is typically required, as the lipid substrate is not water-soluble, and when lipid is presented to the enzyme in the form of an aqueous liposomal dispersion, the enzymatic activity is usually poor. Conventional methods for this reaction have generally employed a two-phase system containing water, in which the enzyme is soluble, and an organic solvent, in which the lipid is soluble, with the enzymatic reaction occurring at the interface between the water and the organic solvent.
The use of detergents or organic solvents is problematic in large scale reactions, and may be prohibited in the production of many pharmaceutical and food products. In addition, some amount of the hydrolysis product, phosphatidic acid (PA), is typically formed in such reactions, and methods are sought for increasing conversion to the transesterification product.